Infectious bursal disease virus (IBDV), a member of the Bimaviridae family, is the causative agent of a highly immunosuppressive disease in young chickens (Kibenge, F. S. B., et al., J. Gen. Virol., 69, 1757-1775 (1988)). Infectious bursal disease (IBD) or Gumboro disease is characterized by the destruction of lymphoid follicles in the bursa of Fabricius. In a fully susceptible chicken flock of 3-6 weeks of age the clinical disease causes severe immunosuppression, and is responsible for losses due to impaired growth, decreased feed efficiency, and death. Susceptible chickens less than 3 weeks old do not exhibit outward clinical signs of the disease but have a marked infection characterized by gross lesions of the bursa.
The virus associated with the symptoms of the disease is called infectious bursal disease virus (IBDV). IBDV is a pathogen of major economic importance to the nation and world""s poultry industries. It causes severe immunodeficiency in young chickens by destruction of precursors of antibody-production B cells in the bursa of Fabricius. Immunosuppression causes increased susceptibility to other diseases, and interferes with effective vaccination against Newcastle disease, Marek""s disease and infectious bronchitis disease viruses.
There are two known serotypes of IBDV. Serotype I viruses are pathogenic to chickens whereas serotype II viruses infect chickens and turkeys. The infection of turkeys is presently of unknown clinical significance.
IBDV belongs to a group of viruses called Bimaviridae which includes other bisegmented RNA viruses such as infectious pancreatic necrosis virus (fish), tellina virus and oyster virus (bivalve mollusks) and drosophila X virus (fruit fly). These viruses all contain high molecular weight (MW) double-stranded RNA genomes.
The capsid of the IBDV virion consists of several structural proteins. As many as nine structural proteins have been reported but there is evidence that some of these may have a precursor-product relationship (Kibenge, F. S. B., et al., J. Gen. Virol., 69, 1757-1775 (1988)). The designation and molecular weights of the viral proteins (VP) are as shown below.
Two segments of double-stranded RNA were identified in the genome of IBDV. The IBDV genome consists of two segments of double-stranded (ds)RNA that vary between 2827 (segment B) to 3261 (segment A) nucleotide base pairs (Mundt, E. et al., Virology, 209, 10-18 (1995)). The larger segment A encodes a polyprotein which is cleaved by autoproteolysis to form mature viral proteins VP2, VP3 and VP4 (Hudson, P. J. et al., Nucleic Acids Res., 14, 5001-5012 (1986)). VP2 and VP3 are the major structural proteins of the virion. VP2 is the major host-protective immunogen of IBDV, and contains the antigenic regions responsible for the induction of neutralizing antibodies (Azad, et al., Virology, 161, 145-152 (1987)). A second open reading frame (ORF), preceding and partially overlapping the polyprotein gene, encodes a, protein (VP5) of unknown function that is present in IBDV-infected cells (Mundt, E., et al., J Gen. Virol., 76, 437-443, (1995)). The smaller segment B encodes VP1, a 90-kDa multifunctional protein with polymerase and capping enzyme activities (Spies, U., et al., Virus Res., 8, 127-140 (1987); Spies, U., et al., J. Gen. Virol., 71, 977-981 (1990)).
It has been demonstrated that the VP2 protein is the major host protective immunogen of IBDV, and that it contains the antigenic region responsible for the induction of neutralizing antibodies. The region containing the neutralization site has been shown to be highly conformation-dependent. The VP3 protein has been considered to be a group-specific antigen because it is recognized by monoclonal antibodies directed against it from strains of both serotype I and II viruses. The VP4 protein appears to be a virus-coded protease that is involved in the processing of a precursor polyprotein of the VP2, VP3 and VP4 proteins.
Although the nucleotide sequences for genome segments A and B of various IBDV strains have been published, it was only recently that the complete 5xe2x80x2- and 3xe2x80x2-noncoding sequences of both segments were determined. The 5xe2x80x2-noncoding region of IBDV segments A and B contain a consensus sequence of 32 nucleotides, whereas the 3xe2x80x2-noncoding terminal sequences of both segments are unrelated, but conserved among IBDV strains of the same serotype (Mundt, E. et al., Virology, 209, 10-18 (1995)). These terminii might contain sequences important in packaging and in the regulation of IBDV gene expression, as demonstrated for other dsRNA containing viruses such as mammalian and plant reoviruses, and rotaviruses (Anzola, et al., Proc. Natl. Acad. Sci. USA, 84, 8301-8305 (1987); Zou, S., et al., Virology, 186, 377-388 (1992); Gorziglia, M. I., et al., Proc. Natl. Acad. Sci. USA, 89, 5784-5788 (1992)).
In recent years, a number of infectious animal RNA viruses have been generated from cloned cDNA using transcripts produced by DNA-dependent RNA polymerase (Boyer, J. C., et al., Virology, 198, 415-426 (1994)). For example poliovirus, a plus-stranded RNA virus; influenza virus, a segmented negative-stranded RNA virus; rabies virus, a non-segmented negative-stranded RNA virus; all were recovered from cloned cDNAs of their respective genomes (van der Werf, S., et al., Proc. Natl. Acad. Sci. USA, 83, 2330-2334 (1986); Enami, M., et al., Proc. Natl. Acad. Sci. USA, 87, 3802-3805 (1990); Schnell, M. J., et al., EMBO J., 13, 41954205 (1994)). For reovirus, it was shown that transfection of cells with a combination of SSRNA, dsRNA and in vitro translated reovirus products generated infectious reovirus when complemented with a helper virus from a different serotype (Roner, M. R., et al., Virology, 179, 845-852 (1990)). However, to date, there has been no report of a recovered infectious virus of segmented dsRNA genome from synthetic RNAs only.
This invention relates to the infectious bursal disease virus (IBDV) that is associated with Gumboro disease of young chickens. More particularly, this invention relates to a system for the generation of infectious bursal disease virus (IBDV) using synthetic transcripts derived from cloned cDNA. The present invention will facilitate studies of the regulation of viral gene expression, pathogenesis and design of a new generation of live and inactivated vaccines.
In an effort to develop a reverse genetics system for IBDV, three independent full-length cDNA clones which contain segment A of serotype I strain D78 or serotype II strain 23/82 and segment B of the serotype I strain P2, respectively, were constructed. Synthetic RNAs of segments A and B were produced by in vitro transcription reaction on linearized plasmids with T7 RNA polymerase. Transcripts of these segments, either untreated or treated with DNase or RNase, were evaluated for the generation of infectious virus by transfection of Vero cells.
The present inventors have demonstrated that synthetic transcripts derived from cloned DNA corresponding to the entire genome of a segmented dsRNA animal virus can give rise to a replicating virus. The recovery of infectious virus after transfecting cells with synthetic plus-sense RNAs derived from cloned cDNA of a virus with a dsRNA genome (IBDV) completes the quest of generating reverse infectious systems for RNA viruses. A number of investigators have generated infectious animal RNA viruses from cloned cDNA (Boyer, J. C., et al., Virology, 198, 415-426 (1994)). Van der Werf et al. were first to generate poliovirus, a plus-stranded RNA virus, using synthetic RNA produced by T7 RNA polymerase on cloned cDNA template (van der Werf, S., et al., Proc. Natl. Acad. Sci. USA, 83, 2330-2334 (1986)). later, Enami et al. rescued influenza virus, a segmented negative-stranded RNA virus (Enami, M., et al., Proc. Natl. Acad. Sci. USA, 87, 3802-3805 (1990)); and Schnell et al. generated rabies virus, a non-segmented negative-stranded RNA virus, from cloned cDNAs of their respective genomes (Schnell, M. J., et al., EMBO J., 13, 4195-4205 (1994)). Roner et al. developed an infectious system for a segmented dsRNA reovirus by transfecting cells with a combination of synthetic ssRNA, dsRNA, in vitro translated reovirus products, and complemented with a helper virus of different serotype (Roner, M. R., et al., Virology, 179, 845-852 (1990)). The resulting virus was discriminated from the helper virus by plaque assay. However, in this system the use of a helper virus was necessary. In contrast, the presently described reverse genetics system of IBDV does not require a helper virus or other viral proteins. Transfection of cells with plus-sense RNAs of both segments was sufficient to generate infectious virus (IBDV). The fate of the additional one or four nucleotides, respectively, transcribed at the 3xe2x80x2-end of segment A was not determined. However, this did not prevent the replication of the viral dsRNA. Similar effects were observed for plus-stranded RNA viruses by different investigators (Boyer, J. C., et al., Virology, 198, 415-426 (1994)).
Transfection of plus-sense RNAs of both segments into the same cell was necessary for the successful recovery of IBDV. Transfected RNAs of both segments had to be translated by the cellular translation machinery. The polyprotein of segment A was presumably processed into VP2, VP3 and VP4 proteins which form the viral capsid. The translated protein VP1 of segment B probably acted as a RNA-dependent RNA polymerase and transcribed minus-strands from synthetic plus-strands of both segments, and the reaction products formed dsRNA. Recently, Dobos reported that in vitro transcription by the virion RNA-dependent RNA polymerase of infectious pancreatic necrosis virus (IPNV), a prototype virus of the Bimaviridae family, is primed by VP1 and then proceeds via an asymmetric, semiconservative, strand-displacement mechanism to synthesize only plus strands during replication of the viral genome (Dobos, P., Virology, 208, 10-25 (1995)). The present system shows that synthesis of minus-strands proceeds on the plus-strands. Whether the resulting transcribed minus-strand RNA serves as a template for the transcription of plus-strands or not remains the subject of further investigation.
To prove that the infectious IBDV contained in the supernatants of transfected cells was indeed derived from the synthetic transcripts, an artificial chimera was generated containing segment A of a serotype II strain and segment B of a serotype I strain. Sequence analysis verified this genome combination. The results also indicate that the terminal sequence motifs described by Mundt and Mxc3xcller are probably responsible for replication, sorting and packaging of the viral genome (Mundt, E. et al., Virology, 209, 10-18 (1995)). Presence of serotype-specific terminal sequences obviously does not prevent proper replication of serotype II A segment by the action of the RNA-dependent RNA polymerase VP1 of the serotype I segment B. The ability to create recombinant viruses will greatly help in analyzing the precise function of serotype-specific and serotype-common terminal sequences.
The recovery of infectious IBDV demonstrates that only the plus-strand RNAs of both segments are sufficient to initiate replication of dsRNA. Thus, the results are in agreement with the general features of reovirus and rotavirus replication where the plus-strand RNAs serve as a template for the synthesis of progeny minus-strands to yield dsRNA (Schonberg, M., et al., Proc. Natl. Acad. Sci. Patton, J. T., Virus Res., 6, 217-233 (1986); Chen, D., et al., J. Virol, 68, 7030-7039 (1994)). However, the semiconservative, strand displacement mechanisms proposed by Spies et al. and Dobos could not be excluded (Spies, U., et al., Virus Res., 8, 127-140 (1987); Dobos, P., Virology, 208, 10-25 (1995)). The development of a reverse genetics system for IBDV will greatly facilitate future studies of gene expression, pathogenesis, and help in the design of new generations of live and inactivated IBDV vaccines.
As used in the present application, the term xe2x80x9csyntheticxe2x80x9d as applied to nucleic acids indicates that it is a man made nucleic acid in contrast to a naturally occurring nucleic acid. The term implies no limitation as to the method of manufacture, which can be chemical or biological as long as the method of manufacture involves the intervention of man.
The term xe2x80x9ccDNAxe2x80x9d is intended to encompass any cDNA containing segments A and B and the 5xe2x80x2 and 3xe2x80x2 noncoding regions of segments A and B.
The term xe2x80x9cinfectiousxe2x80x9d as applied to viruses indicates that the virus has the ability to reproduce. The virus can be pathogenic or nonpathogentic and still be infectious.
The present invention provides a system for the generation of infectious bursal disease virus using synthetic RNA transcripts. This system can be used to study the regulation of viral gene expression, pathogenesis, and for the design of a new generation of live and inactivated IBDV vaccines.
The present invention provides a recombinant vector containing at least one copy of the cDNA according to the present invention. The recombinant vector may also comprise other necessary sequences such as expression control sequences, markers, amplifying genes, signal sequences, promoters, and the like, as is known in the art. Useful vectors for this purpose are plasmids, and viruses such as baculoviruses, herpes virus (HVT) and pox viruses, e.g., fowl pox virus, and the like.
Also provided herein is a host cell transformed with the recombinant vector of the present invention or a host cell transfected with the synthetic RNA of the present invention. The host cell may be a eukaryotic or a prokaryotic host cell. Suitable examples are E. coli, insect cell lines such as Sf-9, chicken embryo fibroblast (CEF) cells, chicken embryo kidney (CEK) cells, African green monkey Vero cells and the like.
Also part of this invention is an IBDV poultry vaccine comprising a poultry protecting amount of a recombinantly produced virus or portion of a virus, wherein the virus is inactivated or modified such that it is no longer virulent.
The virus can be inactivated by chemical or physical means. Chemical inactivation can be achieved by treating the virus with, for example, enzymes, formaldehyde, xcex2-propiolactone, ethylene-imine or a derivative thereof, an organic solvent (e.g. halogenated hydrocarbon) and or a detergent. If necessary, the inactivating substance can be neutralized after the virus has been inactivated. Physical inactivation can be carried out by subjecting the viruses to radiation such as UV light, X-radiation, or xcex3-radiation.
The virus can be attenuated by known methods including serial passage, deleting sequences of nucleic acids and site directed mutagenesis either before or after production of the infectious virus to produce a virus which retains sufficient antigenicity but which has reduced virulence.
Physiologically acceptable carriers for vaccination of poultry are known in the art and need not be further described herein. In addition to being physiologically acceptable to the poultry the carrier must not interfere with the immunological response elicited by the vaccine and/or with the expression of its polypeptide product.
Other additives, such as adjuvants and stabilizers, among others, may also be contained in the vaccine in amounts known in the art. Preferably, adjuvants such as aluminum hydroxide, aluminum phosphate, plant and animal oils, and the like, are administered with the vaccine in amounts sufficient to enhance the immune response to the IBDV. The amount of adjuvant added to the vaccine will vary depending on the nature of the adjuvant, generally ranging from about 0.1 to about 100 times the weight of the IBDV, preferably from about 1 to about 10 times the weight of the IBDV.
The vaccine of the present invention may also contain various stabilizers. Any suitable stabilizer can be used including carbohydrates such as sorbitol, mannitol, starch, sucrose, dextrin, or glucose; proteins such as albumin or casein; and buffers such as alkaline metal phosphate and the like. A stabilizer is particularly advantageous when a dry vaccine preparation is prepared by lyophilization.
The vaccine can be administered by any suitable known method of inoculating poultry including nasally, ophthalmically, by injection, in drinking water, in the feed, by exposure, and the like. Preferably, the vaccine is administered by mass administration techniques such as by placing the vaccine in drinking water or by spraying the animals"" environment. When administered by injection, the vaccines are preferably administered parenterally. Parenteral administration as used herein means administration by intravenous, subcutaneous, intramuscular, or intraperitoneal injection.
The vaccine of the present invention is administered to poultry to prevent IBD anytime before or after hatching. Preferably, the vaccine is administered prior to the time of birth and after the animal is about 6 weeks of age. Poultry is defined to include but not be limited to chickens, roosters, hens, broilers, roasters, breeders, layers, turkeys and ducks.
The vaccine may be provided in a sterile container in unit form or in other amounts. It is preferably stored frozen, below xe2x88x9220xc2x0 C., and more preferably below xe2x88x9270xc2x0 C. It is thawed prior to use, and may be refrozen immediately thereafter. For administration to poultry the recombinantly produced virus may be suspended in a carrier in an amount of about 104 to 107 pfu/ml, and more preferably about 105 to 106 pfu/ml in a carrier such as a saline solution. The inactivated vaccine may contain the antigenic equivalent of 104 to 107 pfu/ml suspended in a carrier. Other carriers may also be utilized as is known in the art. Examples of pharmaceutically acceptable carriers are diluents and inert pharmaceutical carriers known in the art. Preferably, the carrier or diluent is one compatible with the administration of the vaccine by mass administration techniques. However, the carrier or diluent may also be compatible with other administration methods such as injection, eye drops, nose drops, and the like.
The invention also can be used to produce combination vaccines with the IBDV material. The IBDV material can be combined with antigen material of Newcastle Disease Virus Infectious Bronchitis virus, Reo virus, Adeno virus and/or the Marek virus.
The foregoing embodiments of the present invention are further described in the following Examples. However, the present invention is not limited by the Examples, and variations will be apparent to those skilled in the art without departing from the scope of the present invention.